EP1975243B1 - BODIPY aminoacetaldehyde diethyl acetal - Google Patents

BODIPY aminoacetaldehyde diethyl acetal Download PDF

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EP1975243B1
EP1975243B1 EP08010501A EP08010501A EP1975243B1 EP 1975243 B1 EP1975243 B1 EP 1975243B1 EP 08010501 A EP08010501 A EP 08010501A EP 08010501 A EP08010501 A EP 08010501A EP 1975243 B1 EP1975243 B1 EP 1975243B1
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cells
aldh
baaa
cell
ucb
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German (de)
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French (fr)
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EP1975243A1 (en
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Clayton A Smith
Michael Colvin
Robert W. Storms
Susan M. Ludeman
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Duke University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • HSC hematopoietic stem cells
  • PHSC pluripotent hematopoietic stem cells
  • MDR multiple drug resistance
  • Sources of cell populations that are suitable for use include umbilical cord blood, bone marrow, peripheral blood and fetal liver. Any cell population that includes stem cells can be used regardless of tissue origin (e.g., gut, skin muscle, nerve, etc.). While the method can be expected to be applicable to a variety of non-human mammalian cell populations, it is particularly useful in isolating human stem cells from sources including those referenced above.
  • the cell preparations that were recovered were up to 65% CD34 + cells, most of which were CD38 -/dim CD71 -dim .
  • the invention includes within its scope cell preparations that are greater than 50% CD34 + cells, preferably greater than 75% CD34 + cells, more preferably, greater than 90% CD34 + cells.
  • the stem cells isolated have application in a variety of therapies and diagnostic regimens. They are suitable for both transplantation and gene therapy purposes.
  • isolation of stem cells from bone marrow or peripheral blood of patients with cancer can provide for the separation of stem cells from cancer cells. In patients undergoing autologous transplantation, such separation can be used to reduce the chance that cancer cells are returned to the patient.
  • Purified autologous stem cells can be ex vivo expanded to hasten neutrophil, erythroid and platelet engraftment after autologous transplantation. Ex vivo expansion can be effected by growth in defined cytokines, on stromal layers and/or in bioreactors ( Emerson et al, Blood 87:3082 (1996 )). In addition, the incidence of graft failure can be reduced. This is beneficial for cancer patients undergoing autologous transplantation, for patients suffering from auto-immune disorders, and for patients undergoing gene therapy.
  • Gene therapy approaches involving the present cells may involve, isolation of autologous stem cells, exposure of the isolated cells to a gene delivery vector and re-infusion of the modified cells into the patient ( Smith, J. Hematother. 1:155 (1992 )).
  • This approach can involve ex vivo culture or the use of vectors capable of transferring genes into non-dividing cells, thereby rendering ex vivo culture unnecessary.
  • Gene therapy can be useful in treating, for example, congenital diseases, such as sickle cell anemia, in which case the mutant ⁇ -globin gene is replaced or supplemented with either the wild type globin gene or an anti-sickling globin gene.
  • drug resistance genes can be introduced into the stem cells to confer resistance to cytotoxic drugs.
  • Purified allogenic stem cells can be ex vivo expanded to hasten neutrophil, erythroid and platelet engraftment after allogeneic transplantation. In addition, the incidence of graft failure can be reduced. This is likely to be particularly important for recipients of umbilical cord blood transplants where small cell doses limit the success of transplantation.
  • cells can be used as sources of new genes (eg for cytokines and cytokine receptors), including genes important in growth and development.
  • the substrate can be used to identify tumors that may be resistant to cyclophosphamide via up regulation of ALDH activity.
  • cells of the tumor can be contacted with the detectable substrate, e.g., BAAA, and MDR inhibitor under conditions such that the substrate enters the cells and is converted therein to the detectable product.
  • Cells that stain brightly e.g., with BAAA
  • BAAA cyclophosphamide resistant.
  • kits that can be used to prepare the cells.
  • the kits can comprise reagents (e.g., ALDH substrate) that can be used to effect isolation of the stem cells.
  • the kit may include BAAA disposed within a container means.
  • the kit can also include, disposed within a container means, an MDR inhibitor, such as verapamil.
  • the aldehyde dehydrogenase substrate is prepared as BODIPY aminoacetal and lyophilized in 0.5 micromole aliquots. These preparations are stable indefinitely when stored at -20°C.
  • the acetal is then solubilized in DMSO to a final concentration of 5 mM. This solution has been found to be stable at 4°C for up to 1 week.
  • To convert the acetal to an acetaldehyde, aliquots of this solution are brought to a final concentration of 1 N HCl. Under these conditions the acetal has a half life of 15 minutes.
  • BODIPY aminoacetaldehyde BODIPY aminoacetaldehyde
  • PBS Dulbecco's phosphate buffered saline
  • Directly-conjugated fluorescent antibodies directed against CD2 (Leu5; FITC), CD3 (Leu4; PerCP), CD5 (Leu1; PE), CD7 (Leu9; FITC), CD10 (CALLA; FITC), CD11b (Leu15; PE), CD14 (Leu M3; PE), CD19 (Leu12; FITC), CD33 (LeuM9; PE), CD34 (HPCA2; FITC and PE), CD38 (Leu17; PE), CD56 (Leu19; PE) and HLA-DR (FITC) from Becton Dickinson Immunocytometry Systems (BDIS; San Jose, CA) were used.
  • BDIS Becton Dickinson Immunocytometry Systems
  • Anti-CD7 (3A1; PE) and anti-CD45 (KC56; PE) were purchased from Coulter Corporation (Hialeah, FL); anti-CD3 (UCHT1; PE), anti-CD16 (3G8; PE), anti-CD19 (J4.119; PE) as well as the pooled anti-CD34 antibodies (QBEnd10, Immu-133, Immu-134; PE) from Immunotech, Inc. (Westbrook, ME); anti-CD3 (B-B11; FITC) and CD38 (B-A6; FITC) from BioSource International (Camarillo, CA); anti-CD45RA (F8-11-13; PE) from Southern Biotechnology Associates, Inc. (Birmingham, AL); and anti-CDw90 (5E10; PE) from PharMingen, Inc. (San Diego, CA).
  • K562, L1210 and L1210/cpa cells were maintained in suspension in RPMI 1640 media supplemented with 10% Fetal Calf Serum (FCS) and 5 x 10 -5 M ⁇ -mercaptoethanol.
  • Non-agglutinated white blood cells were harvested and residual red cells were hemolysed at 37°C in 0.17 M NH 4 Cl containing 10 mM Tris-HCl, pH 7.2 and 200 mM EDTA.
  • the recovered cells were washed in IMDM containing 2% FCS and mononuclear cells are then purified using Ficoll-Hypaque (1.077 g/ml). When held overnight, the cells were kept on ice in a 4°C refrigerated room in IMDM with 20% FCS.
  • Mononuclear UCB cells were resuspended at 10 6 cells/ml in IMDM containing 2% FCS and were labeled with 1 ⁇ M BAAA for 30 min. When used, verapamil was included at 50 mM. After staining, the cells were washed with ice cold staining media and maintained on ice until their analysis and sorting. The cells were then resuspended in staining media with 10 mg/ml 7-aminoactinomycin D (7AAD) (Molecular Probes; Eugene, OR). For antibody staining to permit multiparameter analyses, the cells were resuspended in staining media (100 ⁇ l) and antibodies were added directly to the cell suspensions.
  • 7AAD 7-aminoactinomycin D
  • the cells were incubated on ice for 20 min. and then washed again in ice cold staining media. The cells were then analyzed or sorted on a FACStar Plus cell sorter (BDIS) equipped with dual Coherent I-90 lasers + an argon-dye laser. The BAAA was excited at 488 nm and emissions were detected using 515 DF20 filter in FL1. Dead and dying cells were excluded on the basis of their high emission in the far red wavelength due to their uptake of 7AAD.
  • BDIS FACStar Plus cell sorter
  • ALDH br cells were isolated directly from mononuclear UCB cells which had been stained with BAAA. For these assays, the ALDH br was defined as 1% of the lymphocyte gate of the UCB.
  • Hematopoietic progenitor colony assays were performed by plating 100-200 cells in MethoCult H4431 containing agar leukocyte conditioned media and recombinant human erythropoietin (StemCell Technologies, Inc.). The cells were incubated in a humidified chamber at 37°C with 5% CO 2 . Hematopoietic colonies (>100 cells) were then scored at 14 to 18 days after initiating the cultures. Long term cultures were maintained on stromal layers of murine MS-5 cells (provided by Dr. Tadashi Sudo of the Kirin Pharmaceutical Research Laboratory, Gunma, Japan) ( Issaad, Blood 81:2916 (1993 )).
  • MS-5 stromal cells were seeded into 24-well plates (Corning Costar Corp., Cambridge, MA) at 5 X 10 4 cells/well in DMEM supplemented with 10% FCS and cultured at 37°C. When the monolayers approached 80% confluence they were ⁇ -irradiated from a cesium source (40 Gy). After irradiation, fresh media was provided to the cultures. For the MS-5 cells, the culture media was replaced entirely with MEM ⁇ supplemented with 10% FCS,10% equine serum, ⁇ -mercaptoethanol, pyruvate. Long term cultures were initiated with 400-2000 hematopoietic progenitor cells/well and were maintained at 33°C with 5% CO 2 .
  • the reagent is prepared and stored as an acetal. Immediately prior to its use, the acetal is converted to an aldehyde in 1 N HCL. the aldehyde is freely soluble in PBS and can be added directly to cells prepared in IMDM with 2% fetal calf serum at 10 6 cells per ml. As an aminoacetaldehyde, the reagent is membrane permeable; however, in the presence of the aldehyde dehydrogenase (ALDH), the aldehyde moiety is converted to a carboxylic acid that is retained in the cell. Intracellular fluorescence can be used to select cells.
  • ADH aldehyde dehydrogenase
  • BAAA is a Specific Substrate for ALDH.
  • ALDH isoenzymes exist and these may display different abilities to convert BAAA. It has been suggested that resistance to cyclophosphamide is primarily mediated by a specific ALDH isoenzyme, ALDH1. Therefore, a human cell line known to express ALDH1, K562, was assayed with this novel reagent. K562 cells converted BAAA and were positive for ALDH in these assays. This response was entirely inhibited by DEAB. Thus, BAAA can serve as a specific substrate for human ALDH1 and can be used to identify primary human cells that demonstrate resistance to cyclophosphamide.
  • Primary UCB Cell preparations contain subsets of ALDH br cells.
  • the BAAA was therefore titrated to an optimal concentration of 1 ⁇ M. This was the best concentration for resolving ALDH br subpopulations.
  • the response was inhibited in the presence of excess DEAB and was therefore specific for ALDH. This molar concentration is 50-fold lower than the concentration of dansyl aminoacetaldehyde that had previously been used to detect murine pluripotent hematopoietic stem cells.
  • the fluorescence emission from BAAA-stained UCB cells exhibited a bimodal response.
  • the brighter peak of fluorescence emission was attributed to mature monocytes, suggesting monocytes express a uniform level of ALDH.
  • Hematopoietic stem cells are small, non-complex cells. Indeed, murine ALDH + PHSC were first enriched using countercurrent elutriation. Therefore, the BODIPY signal was examined only in non-complex cells that exhibited low inherent orthogonal light scattering (SSC lo ) ( Fig. 3A ).
  • the majority of the SSC lo UCB cells were ALDH neg/dim ( Fig. 1B ). This was not unexpected since the SSC lo cells are predominantly lymphocytes, and most lymphocytes do not express ALDH. However, a small, clearly-defined subpopulation of the SSC lo UCB cells was ALDH br ( Fig. 3A ).
  • BODIPY aminoacetate is a substrate for the MDR efflux Pump.
  • PHSC In addition to expressing ALDH, PHSC should also express high levels of the P-glycoprotein or multiple drug resistance (MDR) efflux pump. Since this reagent had never been previously characterized, the susceptibility of BAAA to MDR efflux was assayed. Although BODIPY aminoacetaldehyde passes through the cell membrane without active transport, the product of the ALDH conversion (BODIPY aminoacetate) might well be a substrate for the MDR pump. To investigate this possibility, UCB cells were stained with BAAA in the presence of 50 ⁇ M verapamil, a competitive inhibitor of the MDR efflux pump.
  • MDR multiple drug resistance
  • the verapamil-treated cells exhibited a consistently-higher fluorescence when compared with BAAA-stained cells that had not been simultaneously treated with verapamil ( Fig. 2 ).
  • a substantial population of ALDH dim cells were affected by the verapamil treatment.
  • the percentage of ALDH br cells increased by 1.8 fold in the presence of verapamil.
  • the ALDH br subpopulation was equivalent to 0.8 ⁇ % of the SSC lo cells.
  • the same fluorescence intensity represented only 0.46 ⁇ % of the SSC lo cells. This indicated that the ALDH br SSC lo UCB cells retain the converted BAAA more effectively if the efflux activity of the MDR pump is inhibited.
  • ALDH br SSC lo UCB cells are highly enriched for primitive CD34 + cells .
  • the ALDH br SSC lo UCB cells contained almost 90% CD34 + cells, indicating that at least some hematopoietic progenitors are present ( Fig. 3D ).
  • CD34 is expressed by a broad range of hematopoietic progenitors that includes lineage committed cells, as well as pluripotent progenitors. Therefore, the developmental potential of the ALDH br SSC lo UCB cells was analyzed. Initially, the immunophenotype of these cells was more carefully defined.
  • the immunophenotype would in no way be conclusive; however, the primitiveness of the cell population could be inferred by examining two activation markers that are typically associated with the differentiation of primitive cells to more lineage-committed hematopoietic cells, CD38 and CD71.
  • the most primitive subsets of CD34 + cells have little to no expression of the activation antigens CD38 or CD71.
  • the ALDH br SSC lo UCB cells provided a single-step enrichment for essentially purified CD34 br CD38 dim cells.
  • ALDH expression was inversely proportional to the expression of both CD38 and CD71 ( Fig. 4A and 4B ).
  • the ALDH br SSC lo UCB cells appear to contain the primitive CD34 + cells as defined by immunophenotype.
  • the developmental potential of the ALDH br SSC lo UCB cells were isolated and placed into both short-term and long-term assays for myeloerythroid progenitors.
  • the short-term assay used the hematopoietic progenitor colony assay (HPCA), quantifies lineage committed cells at the time of the initial isolation.
  • More primitive progenitors were also assayed by maintaining the ALDH br SSC lo UCB cells on stroma for either 5 or 8 weeks prior to performing the HPCA ( Fig. 5 ).
  • ALDH br UCB cells have been shown to be predominantely CD34 + CD38 -/lo and highly enriched for early myeloid progenitors. The current study was undertaken to determine whether the ALDH br CD34 + UCB cells were enriched for lymphoid progenitors as well. In 3 experiments, cultures of ALDH br CD34 + UCB cells were established on AFT024 stromal cells in the presence of Kit ligand, Flt3 ligand, IL-3 (1st day only), IL-2 and IL-7 at various dilutions. After 7-8 weeks, the cultures were analyzed for lymphocyte growth as determined by expression of CD56, CD10, CD19 or CD20. Table 1 ALDH br cells/well total wells initiated wells with viable cells lymphocyte + wells 1000 6 5 5 250 16 12 12 62 48 40 40 16 48 34 34 10 24 20 17
  • the AFT024 cultures primarily favored the growth of presumptive NK cells, so to more effectively test whether ALDH br CD34 + UCB cells contained B-lymphoid progenitors, they were cultured on the W20 stromal cell line supplemented with the same cytokine combination. Of 12 cultures established with 100 ALDH br CD34 + cells, all produced CD56 + and CD10 + cells at nearly equivalent proportions. 2 of the 12 wells also contained CD19 + cells.
  • the ALDH br CD34 + UCB population appears to be highly enriched for both myeloid and lymphoid hematopoietic progenitors.

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EP08010501A 1998-12-07 1999-12-07 BODIPY aminoacetaldehyde diethyl acetal Expired - Lifetime EP1975243B1 (en)

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CY20101100522T CY1111098T1 (el) 1998-12-07 2010-06-11 Bodipy διαιθυλοακεταλη αμινοακεταλδεϋδης

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US11119598P 1998-12-07 1998-12-07
EP99965994A EP1137798B1 (en) 1998-12-07 1999-12-07 A method of isolating stem cells

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JP2002531139A (ja) 2002-09-24
DE69942185D1 (de) 2010-05-06
EP1137798A4 (en) 2003-06-25
CY1111042T1 (el) 2015-06-11
US7754480B2 (en) 2010-07-13
DE69939945D1 (de) 2009-01-02
CY1111098T1 (el) 2015-06-11
US20060051833A1 (en) 2006-03-09
US6991897B2 (en) 2006-01-31
CA2353701A1 (en) 2000-06-15
DK1137798T3 (da) 2009-04-14
WO2000034507A1 (en) 2000-06-15
US20040023318A1 (en) 2004-02-05
US6627759B1 (en) 2003-09-30
ATE414783T1 (de) 2008-12-15
ATE462014T1 (de) 2010-04-15
CA2353701C (en) 2014-06-03
DK1975243T3 (da) 2010-06-28
EP1137798B1 (en) 2008-11-19
ES2343222T3 (es) 2010-07-26
EP1137798A1 (en) 2001-10-04
EP1975243A1 (en) 2008-10-01
AU2165200A (en) 2000-06-26
PT1137798E (pt) 2009-02-20
JP4498614B2 (ja) 2010-07-07
AU774566B2 (en) 2004-07-01

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